Pospiviroidae Viroids in Naturally Infected Stone and Pome Fruits In

21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops

Pospiviroidae viroids in naturally infected stone and pome fruits in Greece Kaponi, M.S.1, Luigi, M.2, Barba, M.2, Kyriakopoulou, P.E.I I Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece 2 CRA-PAV, Centro di Ricerca per la Patologia Vegeta le, 00156 Rome, Italy

Abstract Viroid research on pome and stone fruit trees in Greece is important, as it seems that such viroids are widespread in the country and may cause serious diseases. Our research dealt with three Pospiviroidae species infecting pome and stone fruit trees, namely Apple scar skin viroid (ASSVd), Pear blister canker viroid (PBCVd) and Hop stunt viroid (HSVd). Tissue-print hybridization, reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing techniques were successfully used for the detection and identification of these viroids in a large number of pome and stone fruit tree samples from various areas of Greece (Peloponnesus, Macedonia, Thessaly, Attica and Crete). The 58 complete viroid sequences obtained (30 ASSVd, 16 PBCVd and 12 HSVd) were submitted to the Gen Bank. Our results showed the presence of ASSVd in apple, pear, wild apple (Malus sylvestris), wild pear (Pyrus amygdaliformis) and sweet cherry; HSVd in apricot, peach, plum, sweet cherry, bullace plum (Prunus insititia), apple and wild apple; and PBCVd in pear, wild pear, quince, apple and wild apple. This research confirmed previous findings of infection of Hellenic apple, pear and wild pear with ASSVd, pear, wild pear and quince with PBCVd and apricot with HSVd. Our findingsalsorevealedforthefirsttimethenaturalmixedinfectionof appleandwildapplewith (ASSVd+PBCVd+HSVd), of apple and pear with (ASSVd+PBCVd), and of wild apple with (ASSVd+HSVd), as well as the natural infection of Hellenic sweet cherry, peach, bullace plum and plum with HSVd. To our knowledge, this is the first published report of detecting HSVd and PBCVd in infected apple and wild apple, and ASSVd in sweet cherry. Keywords: ASSVd, PBCVd, HSVd, stone fruit, pome fruit, Greece Introduction Apple scar skin viroid (ASSVd), Hop stunt viroid (HSVd) and Pear blister canker viroid (PBCVd) are members of the family Pospiviroidae (Flores et al., 2003) and have been reported to infect wild and cultivated pome and stone fruit trees in Greece (Kyriakopoulou and Hadidi 1998; Amari et al. 2000; Kyriakopoulou et al. 2001; Boubourakas et al. 2006, 2008). The present study has focused on specifying the host range and geographical extent of viroid infections in Greece and obtaining complete nucleotide sequences of the viroids detected in Rosaceous species from various parts of the country. Materials and methods Sample collection: During 2006-2009, 947 field samples of cultivated and wild pome and stone fruit trees with various symptoms (Fig. 1-4) were collected in different regions of Greece (Macedonia, Peloponnesus, Thessaly, Attica and Crete).

Fig. 1 Pear fruit cv `Kontoula' with ASSVd (scar Fig. 2 Wild pear fruit (Pyrus amygdaliformis) with skin, Argolis, Peloponnesus) ASSVd (scar skin, Achaia, Peloponnesus)

Julius-Ktihn-Archiv, 427, 2010 353 21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops

Fig. 3 Pear twig cv `Kontoula' with PBCVd (pear Fig. 4 Apple fruit cv. 'Royal Gala' found to be blister canker, Argolis, Peloponnesus). infected with ASSVd, HSVd and PBCVd (red and white dappling on premature fruit, Pella, Macedonia).

Tissue-print hybridization: Tissue-print hybridization was conducted for all samples, using a modified imprint hybridization protocol of Palacio-Bielsa et al. (1999) and DIG-labelled DNA (ASSVd, PBCVd, HSVd) and RNA (ASSVd) probes. Total RNA extraction and RT-PCR: Total RNA phenol extracts of 120 Rosaceous samples were used in a one tube/two step RT-PCR protocol, employing two different primer pairs per viroid, as described by Faggioli et al. (2001). Cloning and sequencing: Properly-sized RT-PCR products were either sequenced directly or cloned into pGEM-T and pCR® II plasmid vectors, according to the pGEM-T Easy (Promega, Madison, WI, USA ) and TOPO-TA (Invitrogen, Carlsbad, CA, USA) cloning kit instructions, and then sequenced. The sequences obtained were compared with others in the NCBI database and those identified as complete sequence viroid genomes were submitted to the GenBank. Results Tissue print hybridization: ASSVd, PBCVd and HSVd were found in 29.3%, 30.1% and 38.9% of field samples tested by tissue print hybridization, respectively. High frequencies were found for: ASSVd in pear and wild pear from Argolis, Achaia and Corinthia (Peloponnesus), in apple and wild apple trees from Pella (Macedonia) and in sweet cherry trees from Florina (Macedonia). PBCVd was found mainly in pear and wild pear trees from Argolis, Achaia and Corinthia, and in quince trees from Argolis; and HSVd in apricot and peach trees from Argolis and Corinthia, as well as in almond and wild almond (Prunus communis) trees from Achaia, Argolis and Arcadia (Peloponnesus) (Table la). Mixed infections by 2 or 3 viroids, (ASSVd+PBCVd), (ASSVd+HSVd) or (ASSVd+HSVd+PBCVd) were detected in 63 trees from Argolis, Achaia, Corinthia (Peloponnesus) and Pella (Macedonia) (Table lb). Tab. la Viroid-positive fruit tree samples in Greece Viroid Tissue print hybridization RT-PCR Sequences Complete sequences Host species ASSVd PBCVd HSVd ASSVd PBCVd HSVd ASSVd PBCVd HSVd ASSVd PBCVd HSVd 117/400 114/272 232/596 33/120 23/120 39/120 44 21 21 30 16 12 Apple Pella Pella Pella 8 7 4 10 7 3 4 7 2 40% 26% 20% Pear Argolis Argolis 51% 55% Achaia Corinthia 9 8 9 9 5 5 40% 36% Achaia 40% Wild apple Pella Pella Pella (Malta 77% 67% 77% 3 3 3 6 2 5 3 2 3 sylvestris) Wild pear Achaia Argolis (Pyrus 30% 89% amygdaliformis) Corinthia

71% 6 4 3 1 2 1 Achaia 30%

354 Julius-Kiihn-Archiv, 427, 2010 21st International Conference on Virus and other Graft Transmissible Diseases of Fruit Crops

Viroid Tissue print hybridization RT-PCR Sequences Complete sequences Host species ASSVd PBCVd HSVd ASSVd PBCVd HSVd ASSVd PBCVd HSVd ASSVd PBCVd HSVd 117/400 114/272 232/596 33/120 23/120 39/120 44 21 21 30 16 12 Quince Argolis 2 75% Almond Argolis 100% Arcadia 60% Apricot Argolis 60% 11 Corinthia 63% Bullace plum Cherry Florina 6 11 16 5 16 4 100% Peach Argolis 70% 5 2 Corinthia 52% Plum 6 Wild almond Achaia (Prunus 54% 2 communis) Wild plum Corinthia (Prunus spinosa) 80% Tab. lb Viroid in mixed infectious Mixed infections Imprint hybridization Completely sequenced Wild Wild Wild Wild 63 Trees Apple apple Pear pear Quince Cherry Apple apple Pear pear Quince Cherry

ASSVd+PBCVd 10 25 10 2 2 1 2

ASSVd+HSVd 1 6

ASSVd+PBCVd+HSVd 4 6 1 1

RT-PCR: RT-PCR amplified products of the expected size were obtained in 70 out of the 120 field samples tested (Table la). Cloning and sequencing: Cloning and sequencing or direct sequencing (using at least 2 different primers) were completed for 39 RT-PCR products from 31 trees (5 apple, 3 wild apple, 5 pear, 3 wild pear, 1 quince, 7 sweet cherry, 3 apricot, 2 peach, 1 bullace plum (Prunus insititia), 1 Japanese plum), resulting in 44 ASSVd, 21 HSVd and 21 PBCVd sequences. Viroids in mixed infections (ASSVd+HSVd+PBCVd) or (ASSVd+PBCVd) were completely sequenced in 7 trees (Table lb). Fifty-eight complete viroid sequences, 30 ASSVd, 16 PBCVd and 12 HSVd were deposited in the GenBank undertheaccessionnumbersFJ974062-FJ974104,EU925587-EU925591, EU978462-EU978464, GQ249347-GQ249350, GQ141739-GQ141740 and FN376408-FN376409.

AS SVd p heuel .:ins

564 5655E6570 264 334378 374341 342548 12 248 251 filarkrr w. ofrplas - -w. pass - 00 bp: u5P1.2. +.,01.51d

msk =011 4=11 r

Fig. 5 RT-PCR test on 4 apple, 4 pear, 3 wild apple and 3 wild pear total RNA phenol extracts using ASSVd primers. From left: Lane 1, healthy control; lanes 2-15, pome fruit samples; lane 16, positive control; lane 17, Marker 100 bps (Fermentas, LTU).

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Discussion The data obtained in this study indicate that ASSVd, HSVd and PBCVd, previously reported in Greece, are widely spread in cultivated pome and stone fruit trees, including local varieties, especially in areas of great arboricultural importance, such as Central-Western Macedonia and Peloponnesus, as well as wild pears and wild apples and other wild rosaceous species, in the mountains and foothills of the above areas. Our findings also revealed for the first time the natural mixed infection of apple and wild apple with (ASSVd+HSVd+PBCVd), of apple and pear with (ASSVd+PBCVd), and of wild apple with (ASSVd+HSVd), as well as the natural infection of sweet cherry, plum, bullace plum and peach with HSVd in Greece. To our knowledge, this is the first published report of detecting HSVd and PBCVd in apple and wild apple and ASSVd in sweet cherry (Kaponi et al. 2009). Acknowledgements This research project is co-financed by E.U.-European Social Fund (75%) and the Greek Ministry of Development- GSRT (25%). Literature Amari K.; Cariizares M. C.; Myrta A.; Sabanadzovic S.; Srhiri M.; Gavriel I.; Caglayan K.; Varveri C.; Gatt M.; Di Terlizzi B.; Pallas V.; 2000: First report on Hop stunt viroid (HSVd) from some Mediterranean countries. Phytopathologia Mediterranea 39(2), 271-276. Boubourakas I. N.; Hadidi A.; Kyriakopoulou P. E.; 2006: The presence of ASSVd, PBCVd and PLMVd viroids in cultivated and wild pome and stone fruits in Greece. Phytopathologia Mediterranea 45, 173-174. Boubourakas I.N.; Arambatzis C.; Dovas C.; Kyriakopoulou P.E.; 2008: Amelioration of a Reverse Transcription Polymerase Chain Reaction (RT-PCR) for the detection of ASSVd, PBCVd and PLMVd viroids, and their presence in cultivated and wild pome and stone fruits in Greece. Acta Horticulturae 781, 519-527. Faggioli F.; Ragozzino E.; Barba M.; 2001: Simultaneous detection of stone or pome fruit viroids by single tube RT-PCR. Acta Horticulturae 550 (Vol. 1), 59-63. Flores R.; Rand les. J. W.; Owens, R.A; 2003b: Classification. Pages 71-75 in: Viroids, Hadidi A., Flores R., Rand les J. W., Semancik J. S., eds. Collingwood, Australia: CSIRO Publishing. Kaponi M.S., Faggioli F., Luigi M., Barba M., Sano T., and Kyriakopoulou P.E., 2009. First report and molecular analysis of Apple scar skin viroid in sweet cherry. Berichte 21' International Conference on Virus and other Graft-transmissible diseases of Fruit crops (ICVF), Neustadt, Germany, 5-10 July 2009, p. 76 (abstract). Kyriakopoulou P. E.; Giunchedi L.; Hadidi A.; 2001: Peach latent mosaic and pome fruit viroids in naturally infected cultivated pear Pyrus communis and wild pear P. amygdaliformis: Implications on possible origin of these viroids in the Mediterranean region. Journal of Plant Pathology 83(1), 51-62. Kyriakopoulou P. E.; Hadidi A.; 1998: Natural infection of wild and cultivated pears with Apple scar skin viroid in Greece. Acta Horticulturae 472, 617-625. Palacio-Bielsa A.; Foissac X.; Duran-Vila N.; 1999: Indexing of citrus viroids by imprint hybridization. European Journal of Plant Pathology 105, 897-903.

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